Fusion protein-siRNA compound prodrug system responding to tumor microenvironment and application of fusion protein-siRNA compound prodrug system

Abstract

The invention relates to fusion protein, in particular to a tumor microenvironment response fusion protein-siRNA compound prodrug system and application thereof. The vector is a fusion protein formed by coupling a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an RNA binding protein sequence and human heavy chain ferritin. The vector can be combined with siRNA drugs and deliver the siRNA drugs into cells to play a gene silencing role.

Description

Technical Field

[0001] The present invention relates to a fusion protein, and in particular to a tumor microenvironment responsive fusion protein-siRNA complex prodrug system and application thereof. Background Art

[0002] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of about 21 to 23 nucleotides. It is a short molecule formed by the cleavage of double-stranded RNA. It can specifically bind to specific mRNA sequences, leading to the cleavage and degradation of target mRNA, thereby inhibiting gene translation and expression. siRNA targets and binds to specific genes in tumor cells, mediates gene silencing or downregulation, and thus inhibits the growth and proliferation of tumor cells. Compared with traditional chemotherapy, radiotherapy and other treatment methods, siRNA treatment of tumors has many advantages such as high specificity, durability, reversibility, no drug resistance and can be combined with other treatment methods. It is a very promising treatment method.

[0003] The length of siRNA is about 7-8nm, the diameter is about 2-3nm, and the molecular weight is about 13-16kDa. The molecule is too large to pass through the cell membrane, but it is enough to be quickly cleared by glomerular filtration, making it difficult to distribute to the target tissue. siRNA is easily degraded by nucleases that are widely present in the body, and it has high water solubility and polyanionic properties. After intravenous injection, it is quickly filtered and excreted through the kidneys, and the plasma half-life is <10min. Therefore, siRNA must use an effective delivery system.

[0004] siRNA delivery vectors include liposomes, polymers, metal nanoparticles, viral vectors, and extracellular vesicles, which have different advantages and disadvantages. For example, N-acetylgalactosamine (GalNAc) ligands are widely used for siRNA delivery, and GalNAc-siRNA conjugates that exhibit low adverse reactions and limited toxicity have been shown to have high binding affinity to asialoglycoprotein receptors (ASGPRs), which are mainly present on hepatocytes and rarely expressed on other cells. This conjugate-based drug formulation can only treat liver diseases and is prone to degradation and other complications; liposomes are widely used as gene delivery vectors, but liposome-based formulations still have some disadvantages in terms of transfection efficiency, in vivo stability, insufficient targeting ability, toxicity, and immune response; cationic polymers have the problem of high cytotoxicity caused by high positive charges.

[0005] Therefore, finding a new siRNA delivery system is a technical problem that needs to be solved urgently. Among them, the protein carrier delivery siRNA system has significant advantages over the aforementioned delivery carriers: (1) Stability: Proteins can protect siRNA from enzymatic degradation by forming a complex or encapsulating siRNA, thereby improving the stability and half-life of siRNA; (2) Targeting: Proteins have a specific structure and can achieve targeted delivery to specific cells or tissues; (3) Low toxicity: As a natural biological macromolecule, the decomposition metabolites of proteins are generally not toxic to cells and tissues. Compared with chemical synthetics or liposomes, the use of proteins to deliver siRNA is less toxic and can reduce adverse reactions and side effects during treatment; (4) Large-scale preparation: Using genetic recombination technology, protein carriers can be prepared on a large scale, which is scalable and feasible.

[0006] To this end, the present invention provides a new protein carrier system for delivering siRNA. Summary of the invention

[0007] Purpose of the Invention

[0008] The purpose of the present invention is to provide a tumor microenvironment-responsive fusion protein-siRNA complex prodrug system to overcome the defects of the prior art.

[0009] Technical Solution

[0010] A human heavy chain ferritin derivative, characterized in that the sequence from N-terminus to C-terminus comprises a PSTAG shielding sequence as shown in SEQ ID NO: 2, a tumor microenvironment enzyme response sequence as shown in SEQ ID NO: 3, an RNA binding protein as shown in SEQ ID NO: 4, and a human heavy chain ferritin as shown in SEQ ID NO: 5.

[0011] The human heavy chain ferritin derivative is characterized in that the amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.1.

[0012] The human heavy chain ferritin derivative is used in the preparation of a drug delivery carrier.

[0013] A tumor microenvironment-responsive fusion protein-siRNA complex prodrug system, characterized in that the system comprises the human heavy chain ferritin derivative and a small interfering RNA drug with gene silencing effect.

[0014] The system is characterized in that the drug carrier is a nanoparticle formed by the polymerization of human heavy chain ferritin derivatives, and the small interfering RNA drug is combined with the carrier.

[0015] The system is characterized in that the small interfering RNA drug is SEQ ID NO: 6 and SEQ ID NO: 7; or SEQ ID NO: 8 and SEQ ID NO: 9.

[0016] The system is used in the preparation of anti-tumor drugs.

[0017] Specifically:

[0018] The PSTAG 216 (P216), 216 amino acid shielding sequence is

[0019] GPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPSTPAGST, see SEQ ID NO: 2

[0020] The tumor microenvironment MMP2 enzyme response sequence is PLGLAG, see SEQ ID NO: 3;

[0021] The RNA binding protein sequence is

[0022] GDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQVASGVHGPEGFHYKVKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSV, see SEQ ID NO: 4;

[0023] The amino acid sequence of human heavy chain ferritin is

[0024] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES, see SEQ ID NO:5.

[0025] Preferably, the PSTAG shielding sequence, the tumor microenvironment enzyme response sequence, the RNA binding protein and the ferritin are coupled via a GG or GGGGS linker sequence;

[0026] The system is characterized in that the amino acid sequence of the human heavy chain ferritin derivative is:

[0027] , see SEQ ID NO:1.

[0028] Table 1 (Sequence Listing): Description of Fusion Protein Sequences

[0029]

[0030]

[0031] The method for preparing the system is characterized by comprising the following steps: expression and purification of fusion protein, and combination of fusion protein and siRNA to form a complex.

[0032] The system is used to deliver anti-tumor siRNA drugs.

[0033] The application is characterized in that the tumor is liver cancer, the target is the oncogene HBx, and the P216-MMP2-RBP-HFn vector delivers si-HBx for treatment, which has an in vitro anti-tumor effect. The vector delivers siRNA gene therapy drugs, which achieve specific killing of tumors by efficiently targeting and silencing key targets related to tumor growth, metastasis, angiogenesis, drug tolerance and other processes.

[0034] The present invention also relates to a method for preparing the fusion protein-siRNA complex prodrug system. Specifically, the method comprises the following steps:

[0035] Step (1), expressing and purifying the recombinant ferritin derivative

[0036] The full-length sequence of human H-ferritin was obtained by querying the sequences in the NCBI database and references, and PSTAG 216, tumor microenvironment MMP2 enzyme response site, and RNA binding protein sequence were introduced into its N-terminus to design the protein sequence of the fusion protein. After codon optimization with the optimized host being Escherichia coli, the DNA of the fusion protein was constructed into the expression vector pET-28a(+)(GenScript); then, the plasmid of the fusion protein was transformed into the competent expression strain E.coliBL21(DE3) (Vazyme) using the E. coli prokaryotic expression system, and the expression was induced by IPTG. The bacterial solution was collected after fermentation to separate and purify the fusion protein;

[0037] The specific method of expression and purification is as follows: the glycerol inoculated test tube is activated, and after expansion culture in a small conical flask and a small conical flask, it is inoculated in a LB liquid medium containing 1‰ Kan resistance at a volume ratio of 1:100, and 1mM IPTG is added to induce the expression of the target protein under the condition of 37℃ and 220rpm shaking culture. After continuing to culture for 24h, centrifuge at 4℃ and 8000rpm for 15min, discard the supernatant, and collect the bacterial precipitate. The bacterial precipitate is resuspended with 20mM Tris-HCl (pH 8.0) Q column equilibration buffer, and the bacterial lysate is obtained by ultrasonic treatment, and then centrifuged (10000rpm, 20min, 4℃). The supernatant is collected and loaded on the Q column using a peristaltic pump, the flow-through liquid is collected, and the column is rinsed with the Q column equilibration buffer. During this period, the effluent is continuously collected until the G250 does not show blue. Adjust the salt content of Q flow through 20mM Tris to 20mM Tris, 2mM MgCl2, add nuclease (Sinobiological 500Units / g bacteria), incubate at 37℃ for 30min to remove residual nucleic acids in the solution and prevent RBP from binding to other RNAs. Continue to adjust the salt content of Q flow through 20mM Tris, 500mM NaCl (Ni 2+ Column equilibration liquid, use peristaltic pump to load Ni 2+ On the pillar, Ni 2+ The column was washed with column cleaning buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 8.0) and then washed with Ni 2+ Column elution buffer (20 mM Tris, 500 mM NaCl, 200 mM imidazole, pH 8.0) was used to elute the bound proteins. 2+ The target protein is on the column and nucleases without the His tag are removed.

[0038] Samples were taken at each stage, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 12% (v / v) polyacrylamide gel to analyze the size and purity of the protein. The high-purity fractions were collected and placed in a 50kDa dialysis bag for overnight dialysis into 1xPBS buffer. The target protein was concentrated by ultrafiltration using a 50kDa centrifugal ultrafiltration tube to obtain a fusion protein concentrate. Using bovine serum albumin as a standard, the protein concentration was determined using the BCA protein assay reagent (Beyotime). The calculated yield could reach 50mg / L of fermentation broth, which was then packaged and frozen in a -20°C refrigerator.

[0039] Step (2): Use fusion protein to bind siRNA

[0040] DNA templates were designed according to different targeting sequences, and siRNA was transcribed in vitro and purified by centrifugal column (New England Biolabs) according to the in vitro transcription kit (Vazyme).

[0041] The P216-MMP2-RBP-HFn protein was incubated with the corresponding siRNA at 37° C. for 1 h at an N / P (molar ratio of monomeric protein to nucleic acid) ratio of 2 to obtain a complex of the fusion protein and siRNA.

[0042] principle:

[0043] like Figure 7 As shown, the tumor-targeted ferritin derivative carrier of the present invention comprises a fusion protein formed by coupling a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, and an RNA binding protein sequence with human heavy chain ferritin from the N-terminus to the C-terminus. Human heavy chain ferritin self-assembles into a spherical cage-like structure, with the N-terminus of the ferritin subunit facing outward. Only by fusion expression at its N-terminus can other functional proteins be exposed on the outer surface of the globular ferritin.

[0044] Each part realizes the following functions:

[0045] ①PSTAG shielding sequence: prolongs the half-life of the fusion protein, improves the stability of the fusion protein, improves the in vivo distribution of the fusion protein, reduces off-target liver enrichment, and reduces off-target toxicity. ②Tumor microenvironment response site: can be specifically recognized and cut by the highly expressed enzyme MMP2 in the tumor microenvironment, removes the PSTAG shielding sequence, and releases the fusion protein-siRNA complex, RBP-HFn / siRNA, which exerts anti-tumor effects. ③RNA binding protein: can recognize and bind to dsRNA. ④Ferritin: targets tumor cells and exerts intracellular function. The above sequences are connected by flexible Linker GG or EF or GGGGS to allow the proteins on both sides to fold correctly and realize their independent functions.

[0046] The nanoparticles formed by the polymerization of the human heavy chain ferritin derivative, siRNA binds to the RNA binding protein sequence in the carrier to form a fusion protein-siRNA complex prodrug system, which has a strong gene silencing effect, and its mechanism of action is: after the complex enters the body, MMP2 enzyme cleavage occurs in the tumor microenvironment, cutting off the PSTAG shield, exposing RBP-HFn / siRNA, and then TfR1 mediates the introduction into the cell, partially lysosomes escape, and the carrier is degraded and then the siRNA is released to exert a gene silencing effect.

[0047] Beneficial Effects

[0048] RNA binding proteins (RBPs) structurally recognize and interact with siRNA duplexes in a charge-independent manner. The two double-stranded RNA binding domains of protein kinase R (PKR) are able to bind to dsRNA, and this binding is structure-dependent rather than sequence-dependent. dsRBD consists of two dsRNA binding motifs and a highly flexible unstructured linker that allows the two dsRNA binding motifs to be positioned on opposite faces of the dsRNA duplex in a tightly packed manner. The dsRBD protein is the first ribonucleoprotein carrier explored for siRNA delivery.

[0049] Heavy ferritin (HFn) is a spherical cage structure self-assembled from 24 subunits, with an outer diameter of about 12 nm and an inner diameter of about 8 nm. HFn has been used for the delivery of small molecule drugs, metal drugs and nucleic acid drugs due to its unique spherical cage structure, controllable self-assembly, excellent safety, thermal stability, biocompatibility, clear in vivo biological processes and good physicochemical properties. As an endogenous biological carrier, ferritin cage can specifically recognize and bind to transferrin receptor 1 (TfR1) overexpressed on the surface of tumor cells, achieve active targeting, be internalized by tumor cells, and functionally modify ferritin subunits through genetic engineering or chemical coupling. However, there are still problems with HFn carriers. First, TfR1 is significantly expressed in the liver, resulting in HFn being preferentially intercepted by the liver instead of targeting tumors. Secondly, the plasma half-life of HFn is relatively short.

[0050] PSTAG is an unstructured, randomly coiled polypeptide, similar to the chemical PEG polymer. PSTAG was designed and screened as a low immunogenic polypeptide, consisting of five amino acids: Ala, Gly, Pro, Ser, and Thr. It is usually expressed by recombinant fusion with an enzyme-cleavable linker and a therapeutic peptide or protein to increase the half-life and stability of the drug in vivo and mask its activity to form a prodrug system. In tumors, the expression of proteases is often dysregulated, thereby interfering with these physiological processes, which are usually based on the complex interaction between the tumor and the microenvironment. Proteases are upregulated in many tumors and play an important role in the occurrence and development of cancer by promoting extracellular matrix (ECM) remodeling, tumor invasion, and tumor metastasis.

[0051] The invention provides a protein carrier that is stable, safe, efficient, tumor-targeted, and can be used for in vivo delivery of siRNA, namely, the fusion protein PSTAG-MMP2-RBP-HFn. The PSTAG-MMP2-RBP-HFn vector delivers si-HBx to knock down the HBx target in Hep3B cells, achieving an in vitro anti-tumor effect. This provides a new research idea for the in vivo targeted delivery of siRNA into tumor cells. This provides a new research idea for the in vivo targeted delivery of siRNA into tumor cells. It can also provide new technical means for the development of protein intracellular delivery of siRNA.

[0052] The present invention constructs a new fusion protein-siRNA complex prodrug system, which includes a tumor-targeted ferritin derivative carrier and a siRNA gene silencing drug. The tumor-targeted ferritin derivative carrier P216-MMP2-RBP-HFn is a fusion protein formed by coupling a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an RNA binding protein sequence and human heavy chain ferritin. The present invention has the following advantages:

[0053] 1. If Figure 7 As shown, the fusion protein vector is designed as a prodrug structure activatable by tumor microenvironment enzymes, uses a PSTAG shielding sequence, reduces liver accumulation, overcomes the defect of ferritin in the prior art that is preferentially intercepted by the liver rather than targeted to the tumor, and improves safety; prolongs the half-life; the tumor microenvironment enzyme response sequence enhances tumor targeting; ferritin is in a 24-mer form, and one fusion protein expresses 24 RBPs, which can theoretically bind to 24 siRNAs and has a high drug loading capacity.

[0054] 2. If Figure 1 As shown, the fusion protein obtained by prokaryotic expression, separation and purification has high yield (50 mg / L fermentation liquid), high purity (>80%), and is easy to prepare by simple process.

[0055] 3. If Figure 2 As shown, the P216-MMP2-RBP-HFn vector can bind siRNA well to form a complex. Figure 5 As shown, the P216-MMP2-RBP-HFn vector combines with siRNA to form a complex, protects siRNA for delivery, and significantly improves the serum stability and RNase A stability of siRNA.

[0056] 4. If Figure 3 As shown, the P216-MMP2-RBP-HFn vector has a significant intracellular effect and can escape into lysosomes; the vector effectively delivers siRNA into cells, and after RBP-HFn / siRNA escapes into lysosomes, RBP-HFn is degraded to release siRNA.

[0057] 5. If Figure 4 , 5 As shown, the P216-MMP2-RBP-HFn vector delivered siRNA into cells and exerted gene silencing activity, verifying the feasibility of the vector.

[0058] 6. If Figure 5 , 6 As shown, the P216-MMP2-RBP-HFn vector delivered si-HBx, knocked down the oncogene HBx in Hep3B cells, and inhibited the proliferation and migration of liver cancer cells. The P216-MMP2-RBP-HFn vector is an endogenous biological vector that is degradable and has no obvious cytotoxicity.

[0059] 7. If Figure 7 As shown, the fusion protein carrier of the present invention can deliver siRNAs with different targets, increase the anti-tumor effect, and provide a safe, targetable, and easy-to-prepare new protein carrier for anti-tumor siRNA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Preparation and characterization of fusion protein. a is the 12% SDS-PAGE detection of P216-MMP2-RBP-HFn protein purification (lanes: 1: before induction; 2: after induction (25℃ induction); 3: super-top (20mM Tris); 4: super-sinking; 5: Q flow-through; 6: Q elution; 7: Q flow-through (20mM Tris+2mM MgCl2+nuclease 500U / g bacteria 37℃30min); 8: Ni flow-through; 9: Ni 50mM imidazole impurity removal; 10: Ni 200mM imidazole elution); b is the Western of P216-MMP2-RBP-HFn protein Blot identification; c is RP-HPLC analysis of the purity of P216-MMP2-RBP-HFn; d is transmission electron microscopy characterization of P216-MMP2-RBP-HFn; e is SDS-PAGE verification of the temperature stability of P216-MMP2-RBP-HFn; f is the in vitro cleavage verification of MMP2 by P216-MMP2-RBP-HFn.

[0061] Figure 2Preparation and characterization of fusion protein-siRNA complexes. a is a 2% agarose gel electrophoresis of transcribed siRNA (lane: 1: primer annealing (template 1); 2: primer annealing (template 2) 47bp; 3, 4: transcription 21bp; 5, 6: DNaseI, RNaseT1 double enzyme digestion; 7, 8: purification); b is a gel retardation test of P216-MMP2-RBP-HFn / siRNA complexes with different N / P ratios (lane: 1: siRNA; 2: P216-MMP2-RBP-HFn; 3: N / P (monomeric protein / nucleic acid) = 1; 4: N / P = 2);

[0062] Figure 3 The results show that the fusion protein-siRNA complex can enter the cell and escape from the lysosome. a is the flow cytometry test for the cellular uptake efficiency of FAM-P216-MMP2-RBP-HFn; b is the flow cytometry test for the intracellular fluorescence intensity of P216-MMP2-RBP-HFn / Alexa546-siRNA; c is the laser confocal microscopy test for the entry and lysosomal escape of P216-MMP2-RBP-HFn / Alexa 546-siRNA.

[0063] Figure 4 The knockdown effect of EGFP by fusion protein-siRNA complex. a is the change of EGFP level after P216-MMP2-RBP-HFn / si-EGFP treatment detected by flow cytometry; b is the quantitative intracellular mean fluorescence intensity (MFI) by flow cytometry.

[0064] Figure 5 This is a study on the knockdown effect and stability of the fusion protein-siRNA complex in vitro. a is the change in HBx mRNA level detected by RT-qPCR after complex treatment; b is the change in intracellular HBx protein level detected by Western Blot; c is the serum stability of siRNA verified by 2% agarose gel electrophoresis; d is the RNaseA stability of siRNA verified by 2% agarose gel electrophoresis.

[0065] Figure 6 The in vitro anti-tumor effect of fusion protein-siRNA complex. a is the CCK8 assay to detect the changes in the proliferation activity of the administered cells; b is the scratch healing assay to evaluate cell migration; c is the quantitative analysis of the scratch width.

[0066] Figure 7 The schematic diagram of the mechanism of action of the present invention is shown in Figure 1. a is a schematic diagram of the structure of the fusion protein monomer; b is a schematic diagram of P216-MMP2-RBP-HFn binding to siRNA and delivering it into cells to exert gene silencing effect. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0068] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0069] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0070] Example 1 Preparation and characterization of fusion protein

[0071] (1) The full-length sequence of human H-ferritin (HFn) was obtained by querying the sequences in the NCBI database and references. PSTAG 216 (P216), the tumor microenvironment MMP2 enzyme response site (MMP2), and the RNA binding protein sequence (RBP) were introduced into the N-terminus in sequence to design the protein sequence of the fusion protein. After codon optimization with the optimized host being Escherichia coli, the DNA of the fusion protein was constructed into the expression vector pET-28a(+)(GenScript); then, the plasmid of the fusion protein was transformed into the competent expression strain E. coli BL21(DE3) (Vazyme) using the E. coli prokaryotic expression system, and the expression was induced by IPTG. After the bacterial solution was fermented, it was collected and the fusion protein was isolated and purified;

[0072] The specific method of protein expression and purification is as follows: glycerol inoculated test tube activation, after expansion culture in small conical flasks and small conical flasks, inoculated in LB liquid medium containing 1‰ Kan resistance at a volume ratio of 1:100, and 1mM IPTG was added to induce the expression of the target protein under the conditions of 37°C and 220rpm shaking culture. After continuing to culture for 24h, centrifuge at 4°C and 8000rpm for 15min, discard the supernatant, and collect the bacterial precipitate. The bacterial precipitate was resuspended with 20mM Tris-HCl (pH 8.0) Q column equilibration buffer, and the bacterial lysate was obtained by ultrasonic treatment, and then centrifuged (10000rpm, 20min, 4°C). The supernatant was collected and loaded onto the Q column using a peristaltic pump, the flow-through liquid was collected, and the column was rinsed with Q column equilibration buffer. During this period, the effluent was continuously collected until the G250 did not show blue. Adjust the salt content of Q flow through 20mM Tris to 20mM Tris, 2mM MgCl2, add nuclease (Sinobiological 500Units / g bacteria), incubate at 37℃ for 30min to remove residual nucleic acids in the solution and prevent RBP from binding to other RNAs. Continue to adjust the salt content of Q flow through 20mM Tris, 500mM NaCl (Ni 2+ Column equilibration liquid, use peristaltic pump to load Ni 2+ On the pillar, Ni 2+ The column was washed with column cleaning buffer (20 mM Tris, 500 mM NaCl, 50 mM ammonia, pH 8.0) and then Ni 2+ Column elution buffer (20 mM Tris, 500 mM NaCl, 200 mM imidazole, pH 8.0) was used to elute the bound proteins. 2+ The target protein is on the column and nucleases without the His tag are removed.

[0073] Samples were taken at each stage of purification, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 12% (v / v) polyacrylamide gel to analyze the size and purity of the protein. The high-purity fractions were collected and placed in a 50kDa dialysis bag for overnight dialysis into 1xPBS buffer. The target protein was concentrated by ultrafiltration using a 50kDa centrifugal ultrafiltration tube to obtain a fusion protein concentrate. The protein concentration was determined using BCA protein assay reagent (Beyotime) using bovine serum albumin as a standard. The calculated yield could reach 50mg / L fermentation broth, which was then aliquoted and frozen in a -20°C refrigerator.

[0074] (2) Western Blot detection of the purified fusion protein was performed using recombinant Anti-Ferritin HeavyChain antibody (Abcam) for identification. The purified fusion protein was diluted to 0.5 mg / mL with 1×PBS and filtered with a 0.22 μm water filter. The purity of the fusion protein was detected by RP-HPLC using a SepaxGP-C18 column. The column was first rinsed with 5% (v / v) acetonitrile. After the system was stable, the flow rate of the system was set to 1 mL / min, and the detection wavelength was 214 nm. Within 30 minutes, the proportion of mobile phase B was increased from 5% (v / v) to 60% (v / v). The data was recorded and the chromatographic analysis graph was drawn using GraphPad.

[0075] (3) Transmission Electron Microscope (TEM) was used to identify the morphology of the fusion protein. The protein concentration was diluted to 0.5 mg / ml using PBS, filtered using a 0.22 μm filter membrane, and 10 μl was taken for copper mesh sample preparation and photographed.

[0076] (4) To study the stability of the fusion protein, 200 μl of the fusion protein (1 mg / mL) was placed under stable conditions at 37°C and 60°C. At the desired time points (0, 1, 2, 4, 8, 24, 48 h), 20 μl of the sample was taken and quantified by 12% SDS-PAGE.

[0077] (5) To verify that the fusion protein can be cleaved by MMP-2 enzyme, P216 was removed to release RBP-HFn, and the protein cleavage was verified in vitro. 25 μg of protein was cleaved using 1 μl 50 ng / μl MMP-2 pre-activated human (Sigma-Aldrich), incubated at 37°C for 2 h, and samples were taken for 12% SDS-PAGE run.

[0078] result:

[0079] Figure 1 a is the SDS-PAGE identification diagram of the purified fusion protein. After separation and purification, P216-MMP2-RBP-HFn was obtained;

[0080] Figure 1 b is the Western Blot identification of the fusion protein (developed by DAB method), the band size is correct and single;

[0081] Figure 1c is the purity determination of the purified P216-MMP2-RBP-HFn protein by RP-HPLC, and the results show that the peak shape of the P216-MMP2-RBP-HFn protein is symmetrical and the purity is greater than 80%;

[0082] Figure 1 d is the characterization of the fusion protein using a HT7700 microscope (Hitachi, Japan) transmission electron microscope, which shows good dispersion and uniform particle size;

[0083] Figure 1 e is the SDS-PAGE result of the fusion protein after being stored for different time periods. It was found that the fusion protein was very stable at 37°C with little degradation; it could also be stable at 60°C for 8 hours;

[0084] Figure 1 f is the SDS-PAGE characterization of the in vitro cleavage of the fusion protein MMP2. The results show that P216-MMP2-RBP-HFn was successfully cleaved by the MMP2 enzyme to release RBP-HFn.

[0085] Example 2 Preparation and Characterization of Fusion Protein-siRNA Complex

[0086] (1) DNA templates were designed according to different targeting sequences, and siRNA was transcribed in vitro and purified by centrifugal column (New England Biolabs) according to the in vitro transcription kit (Vazyme). The purified siRNA was measured by Nanodrop and stored at -20°C. Samples were taken for identification by 2% agarose gel electrophoresis.

[0087] (2) Verify the binding ability of the fusion protein to siRNA by gel retardation experiment. Prepare 2% agarose gel, and the gel thickness after solidification is 6-8 mm. Incubate P216-MMP2-RBP-HFn protein with the corresponding siRNA at 37°C for 1 h according to the N / P ratio of 0, 1, and 2, where the amount of siRNA used is 1 nmol. After the electrophoresis, use a UV projector to capture the image of the gel.

[0088] (3) According to the experiment, different protein concentrations were selected, and the P216-MMP2-RBP-HFn protein was mixed with the corresponding siRNA at a N / P (molar ratio of monomeric protein to nucleic acid) of 2. The mixture was incubated at 37°C for 1 h to obtain a complex of fusion protein and siRNA.

[0089] result:

[0090] Figure 2 a is the agarose gel electrophoresis verification of in vitro transcribed siRNA, showing the correct band size and high purity;

[0091] Figure 2 b is a gel retardation test of PMRH / siRNA complexes with different N / Ps. The results show that under the condition of N / P of 2, the fusion protein can bind to free siRNA well and achieve a higher drug loading capacity.

[0092] Example 3 Study on the effect of fusion protein-siRNA complex on cell entry and lysosomal escape

[0093] (1) P216-MMP2-RBP-HFn fusion protein was labeled with FAM fluorescent dye. Hep3B cells were plated at 2×10 5 The cells were plated in 24-well plates at a density of 1 μM, 3 μM, and 5 μM FAM-P216-MMP2-RBP-HFn fusion proteins at 37°C for 24 h, digested and collected, and washed three times with 1× PBS buffer solution to remove proteins adsorbed on the cell surface. The cell entry efficiency of the proteins was detected by flow cytometry.

[0094] (2) After treating the cells with free Alexa 546-siRNA, 3 μM P216-MMP2-RBP-HFn, and transfection reagent Lipo2000 (Vazyme) (both protein and Lipo2000 were loaded with equal amounts of Alexa 546-siRNA), flow cytometry was used to detect whether P216-MMP2-RBP-HFn could successfully deliver Alexa 546-siRNA into the cells.

[0095] (3) Add 2×10 5Hep3B cells were cultured for 24 hours. P216-MMP2-RBP-HFn was labeled with FAM, and free FAM was removed by ultrafiltration. 3 μM P216-MMP2-RBP-HFn was mixed with Alexa 546-siRNA at a molar ratio of 2:1 and incubated at 37°C for 1 hour. The culture medium was replaced with OPTI-MEM, and FAM-P216-MMP2-RBP-HFn and P216-MMP2-RBP-HFn / Alexa 546-siRNA were added to the small dishes respectively. After incubation for 24 hours, the cells were rinsed three times with 1×PBS for 5 minutes each time. Lyso-Tracker Red / Green lysosomal tracer (Beyotime) was used to stain cell lysosomes, and then the cells were fixed with formaldehyde and finally the cell nuclei were stained with DAPI. Laser Scanning Confocal Microscopy (LSCM) was used to study the intracellular entry and lysosomal escape of FAM-fusion protein and the intracellular localization of fusion protein / Alexa 546-siRNA, that is, the intracellular entry of Alexa546-siRNA delivered by fusion protein and the lysosomal escape of Alexa 546-siRNA.

[0096] result:

[0097] Figure 3 a is the flow cytometry test of the cellular uptake efficiency of FAM-P216-MMP2-RBP-HFn. The experimental results showed that compared with the Blank group, the flow FITC peaks of the cells in the fusion protein administration group were significantly shifted, indicating that P216-MMP2-RBP-HFn could successfully enter the cell in a concentration-dependent manner;

[0098] Figure 3 b is the flow cytometry test of the cell uptake efficiency of P216-MMP2-RBP-HFn / Alexa 546-siRNA complex. The results showed that the flow PE peak of the complex group cells shifted significantly, indicating that P216-MMP2-RBP-HFn can deliver Alexa 546-siRNA into cells;

[0099] Figure 3 c is the laser confocal microscope to detect the cell entry. The result shows that FAM-P216-MMP2-RBP-HFn can enter the cell well, and the protein is not completely co-localized with the lysosome, indicating that the protein can escape from the lysosome; the fusion protein binds to Alexa546-siRNA and successfully delivers it into the cell. P216-MMP2-RBP-HFn / siRNA escapes from the lysosome, and the fusion protein is degraded by intracellular enzymes and then releases siRNA to exert its effect.

[0100] Example 4: Effect of Fusion Protein-siRNA Complex on Knockdown of EGFP

[0101] A549 cells stably transfected with EGFP were selected as model cells to detect the knockdown efficiency of intracellular EGFP protein expressed by si-EGFP delivered by fusion protein. In the experiment, 3μM P216-MMP2-RBP-HFn was combined with its corresponding si-EGFP at a ratio of N / P=2:1 to form a complex, and co-incubated with A549-EGFP cells in 24-well plates. After 48 hours of administration, flow cytometry was used to detect the silencing efficiency of intracellular EGFP in each administration group to verify the activity of vector-delivered siRNA.

[0102] result:

[0103] Figure 4 a: Changes in EGFP protein levels after treatment with P216-MMP2-RBP-HFn / si-EGFP complex were detected by flow cytometry;

[0104] Figure 4 b is the quantification of intracellular mean fluorescence intensity (MFI), and the results showed that P216-MMP2-RBP-HFn / si-EGFP had a silencing effect on intracellular EGFP, and compared with the Blank group, the EGFP protein level was knocked down by 25%.

[0105] Example 5 In vitro knockdown effect and stability study of fusion protein-siRNA complex

[0106] (1) Hepatitis B virus (HBV) is the main pathogenic factor for the development of liver cancer. The HBV genome encodes the HBx protein. HBx protein is a multifunctional regulatory factor and one of the important factors involved in the HBV-induced hepatocellular carcinogenesis process. It is a potential target for the treatment of HBV-related liver cancer.

[0107] The si-HBx sequence in the reference was used to transcribe si-HBx in vitro. Hep3B liver cancer cells were selected as model cells to detect the knockdown efficiency of HBx protein in Hep3B cells by P216-MMP2-RBP-HFn delivery of si-HBx. In the experiment, 3μM P216-MMP2-RBP-HFn was incubated with si-HBx at a ratio of N / P=2:1 to form a complex, and co-incubated with Hep3B cells in 12-well plates. After incubation for 48h, RNA was extracted, and the knockdown level of HBx by the complex was detected by real-time fluorescence quantitative PCR technology.

[0108] (2) To further identify the silencing efficiency of the complex on intracellular HBx protein expression after P216-MMP2-RBP-HFn / si-HBx was delivered to Hep3B cells, after the drug and cells were incubated for 72 h, the cell proteins in the 6-well plate were extracted, and the difference in intracellular HBx expression was detected by Western Blot (Abcam Anti-Hepatitis B Virus X antigen antibody).

[0109] (3) The serum stability of the P216-MMP2-RBP-HFn / si-HBx complex was studied. 4 nmol of siRNA was incubated with the corresponding amount of P216-MMP2-RBP-HFn (N / P=2) to form a complex. The complex and control siRNA were divided into 4 equal parts (1 nmol), and 10 μl of 100% FBS was added to each part and incubated at 37°C. Samples were taken at 0h, 10h, 18h, and 24h for 2% agarose gel electrophoresis to compare the degree of siRNA degradation in the presence of serum.

[0110] (4) The RNaseA stability of the P216-MMP2-RBP-HFn / si-HBx complex was studied. 3 nmol of siRNA was incubated with the corresponding amount of P216-MMP2-RBP-HFn (N / P=2) to form a complex. The complex and control siRNA were divided into three equal parts (1 nmol), and 1 μl of 0.5 mg / ml RNaseA was added to each part and incubated at 37°C. Samples were taken at 0 h, 1 h, and 2 h for 2% agarose gel electrophoresis to compare the degree of siRNA degradation in the presence of RNaseA.

[0111] result:

[0112] Figure 5 a is the change of HBx mRNA level detected by RT-qPCR after complex treatment. The results showed that compared with the Blank group, P216-MMP2-RBP-HFn / si-HBx treatment of Hep3B cells inhibited HBx mRNA level by 60%;

[0113] Figure 5 b is the change of intracellular HBx protein level detected by Western Blot. The results of Western Blot showed that P216-MMP2-RBP-HFn / si-HBx had a silencing effect on the expression of intracellular HBx;

[0114] Figure 5 c is 2% agarose gel electrophoresis to verify the serum stability of siRNA. The P216-MMP2-RBP-HFn vector has a protective effect on siRNA after binding to it, enhancing its serum stability;

[0115] Figure 5 d is 2% agarose gel electrophoresis to verify the RNase A stability of siRNA. The P216-MMP2-RBP-HFn vector has a protective effect on siRNA after binding to it, enhancing the stability of RNase A.

[0116] Example 6 In vitro anti-tumor effect of fusion protein-siRNA complex

[0117] (1) The effect of the complex on the proliferation of Hep3B cells was detected by CCK8 method. 4 Hep3B cells were plated in 96-well plates. The concentration of the protein was 3 μM, and the N / P ratio of protein to siRNA was 2:1. After 24 hours of administration, 10 μl of CCK8 detection reagent was added to each well and incubated at 37°C for 2 hours. The absorbance (OD) at 450 nm was measured using a Spectra Max I3X microplate reader to calculate the changes in cell proliferation ability.

[0118] (2) A scratch healing experiment was performed to investigate whether the P216-MMP2-RBP-HFn / si-HBx complex could inhibit the migration of Hep3B cells after administration. 5 The cells were evenly spread in a 6-well plate, and the complex was added for incubation after overnight culture. The culture medium was changed to OPTI-MEM and incubated for 24 hours. When the cell culture reached a confluence of about 80%, a 10μl pipette tip was used to vertically scratch the cell monolayer, and the cell debris was washed away with PBS. The culture medium was changed to 1% FBS to reduce the influence of cell proliferation. The cells that migrated to the wound area were monitored using an inverted microscope at 0h, 24h, and 48h. In order to more intuitively display the experimental results, ImageJ was used to perform quantitative analysis of the experimental data.

[0119] result:

[0120] Figure 6 a is the change of cell proliferation activity detected by CCK8 method. The experimental results showed that compared with the Blank group cells, the P216-MMP2-RBP-HFn / si-HBx group inhibited cell proliferation, and the P216-MMP2-RBP-HFn fusion protein had no obvious cytotoxicity;

[0121] Figure 6 b Scratch healing assay to evaluate cell migration. Figure 6c is the quantitative analysis of the scratch healing test. The experimental results showed that compared with the cells in the Blank group, the cells treated with P216-MMP2-RBP-HFn / si-HBx showed a wider wound area 48 hours after wound generation, indicating a lack of migration, which further confirmed that P216-MMP2-RBP-HFn / si-HBx had a silencing efficiency on HBx in Hep3B cells.

Claims

1. A human heavy chain ferritin derivative, characterized in that: The sequence from N-terminus to C-terminus includes a PSTAG shielding sequence as shown in SEQ ID NO: 2, a tumor microenvironment enzyme response sequence as shown in SEQ ID NO: 3, an RNA binding protein as shown in SEQ ID NO: 4, and a human heavy chain ferritin as shown in SEQ ID NO:

5.

2. The human heavy chain ferritin derivative according to claim 1, characterized in that The amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.

1.

3. Use of the human heavy chain ferritin derivative according to claim 1 or 2 in the preparation of a drug delivery carrier.

4. A tumor microenvironment-responsive fusion protein-siRNA complex prodrug system, characterized in that: The system comprises the human heavy chain ferritin derivative according to claim 1 or 2 and a small interfering RNA drug with gene silencing effect.

5. The system according to claim 4, characterized in that The drug carrier is a nanoparticle formed by polymerization of a human heavy chain ferritin derivative, and the small interfering RNA drug is combined with the carrier.

6. The system according to claim 4 or 5, characterized in that: The small interfering RNA drug is SEQ ID NO: 6 and SEQ ID NO: 7; or SEQ ID NO: 8 and SEQ ID NO:

9.

7. Use of the system according to any one of claims 4 to 6 in the preparation of anti-tumor drugs.

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